The present invention relates to the field of controlling pests, such as insects, using a virus to express pest genes in hosts. More specifically, the present invention relates to a method for rapidly screening for pest genes which can lead to mortality of the pest when the pest has ingested host tissues expressing virus-linked pest gene sequences. The present invention also relates to a method for controlling pests by viral expression of target pest sequences to modify endogenous expression of pest genes in cells or tissues of the pest.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the Bibliography.
The earth is full of diverse pest problems and a larger number of methods have been utilized for attempting to control infestations by these pests. Compositions for controlling infestations by microscopic pests such as bacteria, fungi and viruses have been provide in the form of antibiotic compositions, antiviral compositions, and antifungal compositions. Compositions for controlling infestations by larger pests such as nematodes, flatworm, roundworms, pinworms, heartworms, tapeworms, trypanosomes, schistosomes, and the like have typically been in the form of chemical compositions which can either be applied to the surfaces of substrates on which pests are known to infest, or to be ingested by an infested animal in the form of pellets, powders, tablets, pastes, or capsules and the like.
Commercial crops are often the targets of insect attack. Chemical pesticides have been very effective in eradicating pest infestations. However, it is well known that there are several disadvantages to using chemical pesticidal agents. First of all, chemical pesticidal agents are not selective, therefore, on the same time of controlling target insect, because of the lack of selectivity, they also exert their effects on non-target fauna, often effectively sterilizing a field for a period of time over which the pesticidal agents have been applied. Second, chemical pesticidal agents persist in the environment and generally are slow to be metabolized, if at all. They accumulate in the food chain, and finally in the high predator species, such as human being, where these pesticidal agents act as a mutagens and/or carcinogens, to cause irreversible and deleterious genetic modifications. This kind of accumulation causes to higher predator pest resistance. Thus there has been a long felt need for environmentally friendly methods for controlling or eradicating insect infestation on or in plants, i.e., methods which are selective, environmental inset, non-persistent, and biodegradable, and that fit well into pest resistance management schemes. These environmental safe compositions, including Bacillus thuringiensis (Bt) bacteria and transgenic plants expressing one or more genes encoding insecticidal Bt protein, have been remarkably efficient in controlling insect pest infestation. However, with the increased use of Bt crops, such as corn and cotton, comes the threat that target pests may develop resistance to these toxins. Although Bt-resistant insect populations have not yet been observed in the field, resistant strains have been developed in the laboratory by selection with toxin-impregnated diet (McGaughy, 1985). Thus, beside to work out ways to delay Bt resistance development, it is greatly valuable to find a different mode of action to control pest infestations by single use or combined use with Bt expression strategy.
Double stranded RNA (dsRNA) mediated inhibition of specific genes in eukaryotic organisms, has been used to silence genes and study gene function in few insect such as coleopteran Tribolium castaneum (Bucher et al., 2002) previously. Normal delivery of dsRNA to mediate dsRNA-involved genetic control includes generating transgenic insects that express double stranded RNA molecules or injecting dsRNA solutions into the insect body or within the egg sac prior to or during embryonic development. It is widely believed this method of transgenic expression in insect for controlling insect on field crop would be impractical to provide dsRNA molecules in the diet of most invertebrate pest species or to inject compositions containing dsRNA into the bodies of invertebrate pest. Recently, methods using transgenic plants to generate dsRNA have shown that transgenically expressed dsRNA can enhanced resistance to the economically important agricultural pests cotton bollworm (Helicoverpa armigera; Lepidoptera) and Western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte; Coleoptera) (Baum et al., 2007; Mao et al., 2007; U.S. published patent application No 2006/0021087). These references have shown the possibility of using dsRNA in the protection of crops against insect infestation. This approach holds great promise for the future because it allows a wide range of potential targets for suppression of gene expression in the insect to be exploited. However, at this moment, these identified genes are still not as effective as transgenic maize engineered to produce a modified Cry3Bb Bacillus thuringiensis (Bt) toxin.
Therefore the key to compete or even to replace Bt transgenic plant technology is to identify one or more suitable insect genes by feeding expressed dsRNA in vivo in specific plant-insect pair in the content of huge gene number for each agriculturally important pest (for example, 16,404 gene for the model beetle and pest Tribolium castaneum). Previously, one essential technology to evaluate these candidate genes is stable transformation of plants. However, the inefficient production of transgenic plants in some important crops such as cotton limits gene identification on a large scale. Moreover, such procedure is laborious, expensive, time consuming and not suitable for high throughput analysis on a genomic scale.
RNA silencing in plant first was found as a virus resistance as early as 1928 (Wingard, 1928). Wingard described tobacco plants infected with tobacco ringspot virus. The upper leaves had become immune to the virus and consequently were asymptomatic and resistant to secondary infection (Wingard, 1928). Cross protection is therefore widely-used to artificial intervention severe strain virus infection after pre-treated crops with a mild strain in all over the world (Prins et al., 2008). Good examples of diseases control by cross protection were successfully in citrus tristeza and barley yellow dwarf, respectively (Prins et al., 2008).
Infection of plants with both RNA and DNA viruses produces virus-related small interfering RNAs (siRNAs). dsRNA, either derived from a replication intermediate or a secondary-structure characters of some single-stranded viral RNA region, can be accumulated to high levels in virus-infected plant cells. In the case of plant DNA viruses, the dsRNA may be formed by annealing of overlapping complementary transcripts (Baulcombe, 2004). Virus-induced gene silencing for plant gene (VIGS) (Ruiz et al., 1998; Burch-Smith et al., 2004) offers an attractive alternative as it allows the investigation of gene functions without plant transformation in plant gene functional analysis. Recombinant viruses can be constructed carrying an inserted partial sequence of a candidate gene. Such recombinant viruses can move systemically in plants, producing dsRNA (further siRNA) including the inserted fragment of candidate gene that can mediate degradation of the endogenous gene transcripts (Brigneti et al., 2004; Burch-Smith et al., 2004), resulting in silencing of the candidate gene expression in inoculated plants. Depending on the plant species, the effects on endogenous gene expression can usually be assayed 1-2 weeks after virus infection. VIGS can be used as an efficient reverse genetics tool for gene/gene family knock-down in a rapid and high-throughout fashion (Nasir et al., 2005). Because the knock-down phenotype is transient and reversible, this method can be used to access functions of genes whose deficiency may cause embryo lethality (Burch-Smith et al., 2004). Using different injection methods, VIGS has been shown to function in different organs, such as leaves (Liu et al., 2002; Burch-Smith et al., 2006), roots (Valentine et al., 2004; Bhattarai et al., 2007), flowers (Liu et al., 2004; Chen et al., 2005) and even fruits (Fu et al., 2005).
VIGS systems have been successfully applied to assay for gene functions in plants such as Tobacco Rattle Virus in tobacco (Ratcliff et al., 2001), pepper (Chung et al., 2004), tomato (Liu et al., 2002), Jatropha (U.S. Provisional Patent Application No. 61/143,484), cotton (U.S. Provisional Patent Application No. 61/185,631) and poppy (Hileman et al., 2005); Tobacco mosaic virus in tobacco (Hiriart et al., 2003) and pepper (Kim et al., 2007); Potato virus X (PVX) in tobacco (Saitoh and Terauchi, 2002) and potato (Faivre-Rampant et al., 2004); Brome mosaic virus (BMV) in rice, barley and maize (Ding et al., 2006); Barley stripe mosaic virus (BSMV) in barley and wheat (Holzberg et al., 2002); Cucumber mosaic virus in soybean (Nagamatsu et al., 2007); Apple latent spherical virus in tobacco, tomato and soybean (Igarashi et al., 2009; Yamagishi and Yoshikawa, 2009); Bean pod mottle virus in soybean (Zhang and Ghabrial, 2006); Pea early browning virus in Pisum sativum (Constantin et al., 2008), Medicago truncatula and Lathyrus odoratus (Grønlund, et al., 2008); plant DNA virus such as Beet curly top virus (Golenberg et al., 2009) and Tomato yellow leaf curl China virus (Huang et al., 2009). For a general review, see Unver and Budak (2009).
Thus, it is desired to provide alternative and selective means for controlling pest infestation. It is also desired to develop a method for the transient and high-throughput functional analysis of pest genes on a genomic scale to identify pest genes to target for pest control. The present invention provides a method for identifying target pest genes and also provides alternative and selective means for controlling pest infestations.